Poly(A) Binding Protein Is Required for Nuclear Localization of the Ecdysteroidogenic Transcription Factor Molting Defective in the Prothoracic Gland of Drosophila melanogaster

Steroid hormone signaling contributes to the development of multicellular organisms. In insects, ecdysteroids, like ecdysone and the more biologically-active derivative 20-hydroxyecdysone (20E), promote molting and metamorphosis. Ecdysone is biosynthesized in the prothoracic gland (PG), via several steps catalyzed by ecdysteroidogenic enzymes that are encoded by Halloween genes. The spatio-temporal expression pattern of ecdysteroidogenic genes is strictly controlled, resulting in a proper fluctuation of the 20E titer during insect development. However, their transcriptional regulatory mechanism is still elusive. A previous study has found that the polyadenylated tail [poly(A)] deadenylation complex, called Carbon catabolite repressor 4-Negative on TATA (CCR4-NOT) regulates the expression of spookier (spok), which encodes one of the ecdysteroidogenic enzymes in the fruit fly Drosophila melanogaster. Based on this finding, we speculated whether any other poly(A)-related protein also regulates spok expression. In this study, we reported that poly(A) binding protein (Pabp) is involved in spok expression by regulating nuclear localization of the transcription factor molting defective (Mld). When pabp was knocked down specifically in the PG by transgenic RNAi, both spok mRNA and Spok protein levels were significantly reduced. In addition, the spok promoter-driven green fluorescence protein (GFP) signal was also reduced in the pabp-RNAi PG, suggesting that Pabp is involved in the transcriptional regulation of spok. We next examined which transcription factors are responsible for Pabp-dependent transcriptional regulation. Among the transcription factors acting in the PG, we primarily focused on the zinc-finger transcription factor Mld, as Mld is essential for spok transcription. Mld was localized in the nucleus of the control PG cells, while Mld abnormally accumulated in the cytoplasm of pabp-RNAi PG cells. In contrast, pabp-RNAi did not affect the nuclear localization of other transcription factors, including ventral vein lacking (Vvl) and POU domain motif 3 (Pdm3), in PG cells. From these results, we propose that Pabp regulates subcellular localization in the PG, specifically of the transcription factor Mld, in the context of ecdysone biosynthesis.

Steroid hormone signaling contributes to the development of multicellular organisms. In insects, ecdysteroids, like ecdysone and the more biologically-active derivative 20-hydroxyecdysone (20E), promote molting and metamorphosis. Ecdysone is biosynthesized in the prothoracic gland (PG), via several steps catalyzed by ecdysteroidogenic enzymes that are encoded by Halloween genes. The spatio-temporal expression pattern of ecdysteroidogenic genes is strictly controlled, resulting in a proper fluctuation of the 20E titer during insect development. However, their transcriptional regulatory mechanism is still elusive. A previous study has found that the polyadenylated tail [poly(A)] deadenylation complex, called Carbon catabolite repressor 4-Negative on TATA (CCR4-NOT) regulates the expression of spookier (spok), which encodes one of the ecdysteroidogenic enzymes in the fruit fly Drosophila melanogaster. Based on this finding, we speculated whether any other poly(A)-related protein also regulates spok expression. In this study, we reported that poly(A) binding protein (Pabp) is involved in spok expression by regulating nuclear localization of the transcription factor molting defective (Mld). When pabp was knocked down specifically in the PG by transgenic RNAi, both spok mRNA and Spok protein levels were significantly reduced. In addition, the spok promoter-driven green fluorescence protein (GFP) signal was also reduced in the pabp-RNAi PG, suggesting that Pabp is involved in the transcriptional regulation of spok. We next examined which transcription factors are responsible for Pabp-dependent transcriptional regulation. Among the transcription factors acting in the PG, we primarily focused on the zinc-finger transcription factor Mld, as Mld is essential for spok transcription. Mld was localized in the nucleus of the control PG cells, while Mld abnormally accumulated in the cytoplasm

INTRODUCTION
Ecdysteroids, like ecdysone and the more biologically-active derivative 20-hydroxyecdysone (20E), regulate several biological events in insects (Niwa and Niwa, 2014;Uryu et al., 2015). The 20-hydroxyecdysone titers are temporally changed during insect development, where proper fluctuations of 20E titers are essential for insect development (Riddiford, 1993;Nijhout, 1994;Bellés, 2020). Ecdyone is biosynthesized in an endocrine organ named the prothoracic gland (PG) via several steps catalyzed by step-specific enzymes (Niwa and Niwa, 2014). The enzymes are encoded by a group of genes often called the Halloween gene (Rewitz et al., 2007). Therefore, the regulation of ecdysteroidogenic genes expression is essential to achieve proper fluctuations of 20E titers (Niwa and Niwa, 2016). However, the molecular mechanism by which the expression of ecdysteroidogenic genes is regulated is yet to be fully elucidated.
Previously, we have reported that polyadenylated tail [poly(A)] degradation complex, called Carbon catabolite repressor 4-Negative on TATA (CCR4-NOT) is involved in the regulation of ecdysteroidogenic gene expression in the fruit fly Drosophila melanogaster (Zeng et al., 2018). By knocking down the gene phosphoglycerate kinase promoter directed over production (Pop2), which encodes a vital component of the CCR4-NOT complex, specifically in the PG, D. melanogaster animals show a larval-arrested phenotype. Furthermore, the expression levels of some ecdysteroidogenic genes are strongly decreased in these animals. Based on this finding, we hypothesized that other poly(A) related protein(s) may also contribute to the expression of ecdysteroidogenic genes.
In this study, we revealed that poly(A) binding protein (Pabp) contributes to the expression of ecdysteroidogenic genes. The knockdown of the pabp gene in the PG caused first instar-arrest and a decrease in ecdysteroidogenic gene expression, especially spookier (spok). Interestingly, the nuclear localization of the transcription factor molting defective (Mld), a transcriptional activator of spok, was disrupted in PG cells of pabp-RNAi larvae. Our results suggest that Pabp positively regulates ecdysteroidogenic gene expression by regulating ecdysteroidogenic transcription factors.
of pabp-RNAi PG cells. In contrast, pabp-RNAi did not affect the nuclear localization of other transcription factors, including ventral vein lacking (Vvl) and POU domain motif 3 (Pdm3), in PG cells. From these results, we propose that Pabp regulates subcellular localization in the PG, specifically of the transcription factor Mld, in the context of ecdysone biosynthesis.

Quantitative Reverse Transcription-Polymerase Chain Reaction
RNA was isolated from the whole bodies of the second instar larvae using the RNAiso Plus reagent (TaKaRa, Shiga, Japan). Genomic DNA digestion and cDNA synthesis were performed using the ReverTra Ace qPCR RT Kit (TOYOBO, Tokyo, Japan). Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed using the THUNDERBIRD SYBR qPCR Mix (TOYOBO, Tokyo, Japan) with a Thermal Cycler Dic TP800 System (TaKaRa, Shiga, Japan). Serial dilutions of a plasmid containing the open reading frame (ORF) of each gene were used as a standard. The expression levels of the target genes were normalized to an endogenous control ribosomal protein 49 (rp49) in the same sample. Primers amplifying noppera-bo (nobo), neverland (nvd), shroud (sro), spok, phm, disembodied (dib), shadow (sad), and rp49 have been described previously (McBrayer et al., 2007;Niwa et al., 2010;Enya et al., 2014).

GFP Reporter Assay
A spok promoter-driven GFP reporter assay was performed as described previously (Komura-Kawa et al., 2015). Briefly, spok>GFP/CyO Act-GFP; UAS-deicer2, phm22-GAL4/TM6 Ubi-GFP was established and crossed with UAS-pabp-IR. Eggs were laid on grape plates with yeast pastes at 25°C for 2 h. The GFP-negative first instar larvae were picked up and transferred into vials with standard cornmeal food. They were dissected 60 h after egg laying (AEL) and then immunostained.

Pabp Plays an Essential Role in the PG During Larval Development
To examine the importance of Pabp in the PG, we observed the developmental progress of pabp-RNAi larvae, for which we used a PG-specific driver (phm22-GAL4, hereafter phm>) to knock down pabp expression by transgenic RNAi. We found that PG-specific pabp-RNAi caused a larval-arrest phenotype. Eighty eight percent of phm>pabp-RNAi animals were arrested at the second larval instar and even 132 h AEL or later, while only few animals molted into the third larval instar or pupariated at the same time point (Figures 1A-C). The arrested second instar larvae failed to pupariate and died. This result suggests that the Pabp function in the PG is essential for larval development.

PG-Specific Knockdown of pabp Strongly Reduces the Expression of Ecdysteroidogenic Enzyme Genes, Especially spookier
Next, we examined whether pabp knockdown in the PG changes the expression of ecdysteroidogenic genes. We conducted qRT-PCR to examine the expression levels of seven ecdysteroidogenic genes (Chávez et al., 2000;Warren et al., 2002Warren et al., , 2004Niwa et al., 2004Niwa et al., , 2010Namiki et al., 2005;Ono et al., 2006;Yoshiyama et al., 2006;Chanut-delalande et al., 2014;Enya et al., 2014) in the second instar larvae of control and pabp-RNAi animals. The expression levels of all ecdysteroidogenic genes examined were suppressed in pabp-RNAi animals. However, specifically, the suppression levels were different among these seven genes. In particular, the suppression level of spok, encoding an ecdysteroidogenic cytochrome P450 enzyme, substantially decreased (Figure 2A). Consistent with this result, the Spok protein level also substantially decreased in the PG of pabp-RNAi animals as compared to control animals, while the protein level of another ecdysteroidogenic P450 enzyme Phm was only slightly affected (Figures 2B,C).
To determine whether spok expression is transcriptionally and/or translationally disrupted in the pabp-RNAi animals, we conducted a GFP reporter assay in vivo. The spok enhancer region, which is sufficient for spok transcription in the PG, has been identified in our previous study (Komura-Kawa et al., 2015). We found that the spok enhancer-driven GFP level was FIGURE 2 | Expression analysis of ecdysteroidogenic genes. All photos are single-plane confocal images. (A) Relative expression levels of seven ecdysteroidogenic genes in phm>dicer2, pabp-IR at 60 h AEL compared to controls (phm>dicer2, +) based on the quantitative reverse transcription-polymerase chain reaction (qRT-PCR; N = 4). RNA was isolated from the whole bodies of the second instar larvae. (B) Immunostaining of the PG cells from phm>dicer2, + and phm>dicer2, and pabp-IR second instar larvae at 60 h AEL with antibodies against Phm (magenta) and Spok (green). (C) Quantifications of fluorescence intensities of Phm and Spok in the PG (each N = 3). Error bars represent standard deviations. *** and n.s. indicate p < 0.001 and non-significance (p > 0.05), respectively, by Student's t-test. (D) Fluorescence images of the PG cells from phm>dicer2, + and phm>dicer2, pabp-IR larvae with spok enhancer/promoter-driven nuclear localized-GFP construct (spok>GFP) at 60 h AEL. (E) Quantification of GFP fluorescence intensity in the PG nuclei (N = 4). The bar plots are drawn in the same manner as (C). PG cells are immunostained with anti-Phm antibody (magenta). Scale bar: 20 μm.

Reduction of the Expression of spok Correlates With Mislocalization of Its Transcriptional Activator Mld
To analyze the molecular mechanism of suppression of spok transcription, we focused on the transcription factor Mld. Mld is a zinc-finger type DNA binding protein involved in ecdysone biosynthesis in the PG (Neubueser et al., 2005). Moreover, we have previously reported that Mld is crucial for transcription of spok, acting on the upstream region of the spok gene locus (Danielsen et al., 2014;Komura-Kawa et al., 2015). In this study, we newly-generated an anti-Mld antibody to visualize Mld protein in vivo by immunostaining. Before the immunostaining experiment, we examined the specificity of the newly-generated anti-Mld antibody. We observed that the Mld signal was observed in the nucleus of PG cells in control animals. In contrast, the Mld signal in the PG nucleus disappeared in mld-RNAi animals (Figure 3), confirming that the immunostaining signal in the PG nucleus corresponds to Mld protein localization. We then conducted immunostaining with an anti-Mld antibody against pabp-RNAi PG cells. Surprisingly, we found that nuclear localization of Mld was disrupted in the pabp-RNAi PG cells, although Mld is localized in the nucleus of control PG cells (Figures 4A,B). These results suggest that Pabp regulates the transcription of spok by mediating the nuclear localization of Mld. Next, we examined whether such mislocalization is selective for Mld, but not for other transcription factors in PG cells. To address this issue, we examined the nuclear localization of two other transcription factors such as Vvl and Pdm3, in the pabp-RNAi PG cells. Vvl is a POU-domain transcription factor involved in the regulation of the transcription of all known ecdysteroidogenic genes in PG cells (Danielsen et al., 2016). Pdm3 is also a POU-domain transcription factor that is enriched in PG cells (Ou et al., 2016), whereas, its role in PG cells has not yet been elucidated. An immunohistological analysis using anti-Vvl and anti-Pdm3 antibodies revealed that nuclear localization of Vvl and Pdm3 was maintained in the nucleus, even in pabp-RNAi PG cells. Nevertheless, the Vvl signal did slightly decrease (Figures 4C-F). Taken together, these results suggest that Pabp regulates the nuclear localization specifically of Mld.

DISCUSSION
In this study, we revealed that Pabp is required for the nuclear localization of the ecdysteroidogenic transcription factor Mld. First, the PG-specific knockdown of pabp reduced ecdysteroidogenic gene expression, especially that of spok. Second, that reduction of spok expression correlated well with the mislocalization of its transcription factor Mld. Third, mislocalization did not occur for all transcription factors but did occur specifically for Mld. In conjunction with our previous data showing that Mld is crucial for inducing spok expression through the Mld-response element in spok promoter region (Uryu et al., 2018), we propose that Pabp positively regulates spok expression via mediating nuclear localization of Mld in PG cells (Figure 5).
The predicted ORF of mld encodes a protein that belongs to the family of the zinc-finger associated domain (ZAD) containing C 2 H 2 zinc-finger proteins (ZFPs; Neubueser et al., 2005). A previous study has shown that modification or depletion of ZAD disrupts nuclear localization of ZAD-ZFPs (Zolotarev et al., 2016). In conjunction with our observation, this fact raises the possibility that ZAD may be involved in the Pabp-dependent nuclear localization of ZAD-ZFPs, whereas, this hypothesis has yet been experimentally examined. It would also be intriguing to examine whether the subcellular localization of Ouija board (Ouib) and Séance (Séan), other ZAD-ZFP transcription factors of spok and nvd, respectively (Komura-Kawa et al., 2015;Uryu et al., 2018), are also affected by pabp-RNAi. Currently, we failed to generate anti-Ouib and anti-Séan specific antibodies. This is the first report showing a novel function of Pabp in controlling the nuclear localization of a transcription factor for ecdysone biosynthesis. In the last decade, several transcription factors for ecdysone biosynthesis have been identified (Niwa and Niwa, 2016), while the regulation of subcellular localization of these transcription factors has not been rigorously studied; except DHR4 (Ou et al., 2011). Pabp and other poly(A)-related proteins might be critical research targets for understanding the nuclear and cytoplasmic translocation of ecdysteroidogenic transcription factors in the future.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.

FUNDING
This work was supported by a grant from AMED-CREST, AMED to RN (19gm1110001h0003) and the program of the Joint Usage/ Research Center for Developmental Medicine, Institute of Molecular Embryology and Genetics, Kumamoto University to RN. We received for an open access publication fee from our institution.